India ink injection procedure for tapeworm proglottids
Qualitative test for fecal fat
Quantitation of reducing substances (Clinitest)
Among the diagnostic techniques used with stool specimens, the routine ova and parasite (O&P) examination is the best known. This technique has three components: the direct wet mount, the examination of material from a stool concentrate, and the permanent stained smear. This is an excellent procedure and is recommended for most intestinal parasites. However, several other diagnostic techniques are available for the recovery and identification of parasitic organisms. Most laboratories do not routinely offer all of these techniques, but many are relatively simple and inexpensive to perform (1–4). The clinician should be aware of the possibilities and the clinical relevance of information obtained from using such techniques. Occasionally, it is necessary to examine stool specimens for the presence of scolices and proglottids of cestodes and adult nematodes and trematodes to confirm the diagnosis and/or for species identification. A method for the recovery of these stages is also described in this chapter. Although not routinely performed, tests for fecal fat and reducing substances are also included.
Culture of Larval-Stage Nematodes
Nematode infections giving rise to larval stages that hatch in soil or in tissues may be diagnosed by using certain fecal culture methods to concentrate the larvae. Strongyloides stercoralis larvae are generally the most common larvae found in stool specimens. However, depending on the fecal transit time through the intestine and the patient’s condition, rhabditiform and, rarely, filariform larvae may be present. Also, if there is a delay in examination of the stool, embryonated ova as well as larvae of hookworm may be present. Culture of feces for larvae is useful to (i) reveal their presence when they are too scanty to be detected by concentration methods; (ii) distinguish whether the infection is due to S. stercoralis or hookworm on the basis of rhabditiform larval morphology by allowing hookworm egg hatching to occur, releasing first-stage larvae; and (iii) allow the development of larvae into the filariform stage for further differentiation.
The use of certain fecal culture methods (sometimes referred to as coproculture) is especially helpful for detection of light infections with hookworm, S. stercoralis, and Trichostrongylus spp. and for specific identification of parasites. The rearing of infective-stage nematode larvae also helps in the specific diagnosis of hookworm and trichostrongyle infections because the eggs of many of these species are identical and specific identifications are based on larval morphology. Additionally, such techniques are useful for obtaining a large number of infective-stage larvae for research purposes. Four culture techniques and one “enhanced-recovery” method are described in this chapter.
Harada-Mori Filter Paper Strip Culture
To detect light infections with hookworm, S. stercoralis, and Trichostrongylus spp., as well as to facilitate specific identification, the Harada-Mori filter paper strip culture technique is very useful (Fig. 4.1). This filter paper test tube culture technique was initially introduced by Harada and Mori in 1955 (5) and was later modified by others (6, 7).
Figure 4.1 Culture methods for the recovery of larval-stage nematodes: Harada-Mori tube method and petri dish culture method. Viable larvae are present if the specimen contains S. stercoralis or other nematodes. Wear gloves when handling the culture devices. (Illustration by Nobuko Kitamura.) doi:10.1128/9781555819002.ch4.f1
In a study looking at the prevalence of S. stercoralis in three areas of Brazil, the diagnostic efficacy of the agar plate culture method (discussed later in this chapter) was as high as 94.9% compared to only 28.5 and 26.5% by the Harada-Mori filter paper culture and fecal concentration methods, respectively, when fecal specimens were processed using all three methods (8). Among the 49 positive samples, about 60% were confirmed as positive by using the agar plate method alone. These results indicate that the agar plate approach is probably a much more sensitive diagnostic method than the other two and is recommended for the diagnosis of strongyloidiasis.
The technique requires filter paper to which fresh fecal material is added and a test tube into which the filter paper is inserted. Moisture is provided by adding water to the tube, which continuously soaks the filter paper by capillary action. Incubation under suitable conditions favors hatching of ova and/or development of larvae. Fecal specimens to be cultured should not be refrigerated, since some parasites (especially Necator americanus) are susceptible to cold and may fail to develop after refrigeration. Also, caution must be exercised in handling the filter paper strip itself, since infective Strongyloides larvae may migrate upward as well as downward on the paper strip. Always observe standard precautions and wear gloves when performing these procedures.
Quality Control for Harada-Mori Filter Paper Strip Culture
1. Follow routine procedures for optimal collection and handling of fresh fecal specimens for parasitologic examination.
2. Examine known positive and negative samples of stools (from laboratory animals), if available, to gain some experience in using the procedure.
3. Review larval diagrams and descriptions for confirmation of larval identification.
4. The microscope should be calibrated, and the objectives and oculars used for the calibration procedure should be used for all measurements on the microscope. If the microscope undergoes hard use or is moved around within the laboratory, it is strongly recommended that recalibration be performed every 12 months. However, if the microscope remains in the same location and receives normal use, such frequent recalibration may not be required. The calibration factors for all objectives should be posted on the microscope for easy access (multiplication factors can be pasted on the body of the microscope).
5. Record all quality control (QC) results.
Procedure for Harada-Mori Filter Paper Strip Culture
1. Smear 0.5 to 1 g of fresh feces in the center of a narrow strip of filter paper (3/8 by 5 in. [1 in. = 2.54 cm], slightly tapered at one end).
2. Add 3 to 4 ml of distilled water to a 15-ml conical centrifuge tube; identify the specimen on the tube.
3. Insert the filter paper strip into the tube so that the tapered end is near the bottom of the tube. The water level should be approximately 1/2 in. below the fecal spot. It is not necessary to cap the tube. However, a cork stopper or a cotton plug may be used.
4. Maintain the tube upright in a rack at 25 to 28°C. Add distilled water to maintain the original level (usually evaporation takes place over the first 2 days, and then the culture becomes stabilized).
5. Keep the tube for 10 days,
